Citrus sinensis CBF1 Functions in Cold Tolerance by Modulating Putrescine Biosynthesis through Regulation of Arginine Decarboxylase.

C-repeat (CRT) binding factors (CBFs) are well known to act as crucial transcription factors that function in cold stress response. Arginine decarboxylase (ADC)- mediated putrescine (Put) biosynthesis has been reported to be activated in plants exposed to cold conditions, but it remains elusive whether CBFs can regulate ADC expression and Put accumulation. In this study, we show that cold upregulated ADC gene (Citrus sinensis ADC;CsADC) and elevated endogenous Put content in sweet orange (C.sinensis). The promoter of CsADC contains two CRT sequences that are canonical elements recognized by CBFs. Sweet orange genome contains four CBFs (CsCBF1-4), in which CsCBF1 was significantly induced by cold. CsCBF1, located in the nucleus, was demonstrated to bind directly and specifically to the promoter of CsADC and acted as a transcriptional activator. Overexpression of CsCBF1 led to notable elevation of CsADC and Put levels in sweet orange transgenic plants, along with remarkably enhanced cold tolerance, relative to the wild type. However, pretreatment with D-arginine, an ADC inhibitor, caused a prominent reduction of endogenous Put levels in the overexpressing lines, accompanied by greatly compromised cold tolerance. Taken together, these results demonstrate that the CBF1 of sweet orange directly regulates ADC expression and modulates Put synthesis for orchestrating the cold tolerance. Our findings shed light on the transcriptional regulation of Put accumulation through targeting the ADC gene in the presence of cold stress. Meanwhile, this study illustrates a new mechanism underlying the CBF-mediated cold stress response.

Putrescine biosynthetic pathways modulate root growth differently in tomato seedlings grown under different N sources.

The biosynthesis of putrescine is mainly driven by arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). Hence, in this study, we generated independent ADC and ODC transgenic silenced tomato lines (SilADC and SilODC, respectively) to test the effect of defective ADC and ODC gene expression on root development under nitrate (NN) or ammonium (NA) conditions. The results showed that SilODC seedlings displayed an increase in ADC expression that led to polyamine accumulation, suggesting a compensatory effect of ADC. However, this effect was not observed in SilADC seedlings. These pathways are involved in different growth processes. The SilADC seedlings showed an increase in fresh weight, shoot length, lateral root number and shoot:root ratio under the NN source and an enhancement in fresh weight, and shoot and root length under NA conditions. However, SilODC seedlings displayed greater weight and shoot length under the NN source, whereas a decrease in lateral root density was found under NA conditions. Moreover, two overexpressed ODC lines were generated to check the relevance of the compensatory effect of the ADC pathway when ODC was silenced. These overexpressed lines showed not only an enhancement of almost all the studied growth parameters under both N sources but also an amelioration of ammonium syndrome under NA conditions. Together, these results reflect the importance of both pathways in plant growth, particularly ODC silencing, which requires compensation by ADC induction.

Metformin Inhibits the Urea Cycle and Reduces Putrescine Generation in Colorectal Cancer Cell Lines.

The urea cycle (UC) removes the excess nitrogen and ammonia generated by nitrogen-containing compound composites or protein breakdown in the human body. Research has shown that changes in UC enzymes are not only related to tumorigenesis and tumor development but also associated with poor survival in hepatocellular, breast, and colorectal cancers (CRC), etc. Cytoplasmic ornithine, the intermediate product of the urea cycle, is a specific substrate for ornithine decarboxylase (ODC, also known as ODC1) for the production of putrescine and is required for tumor growth. Polyamines (spermidine, spermine, and their precursor putrescine) play central roles in more than half of the steps of colorectal tumorigenesis. Given the close connection between polyamines and cancer, the regulation of polyamine metabolic pathways has attracted attention regarding the mechanisms of action of chemical drugs used to prevent CRC, as the drug most widely used for treating type 2 diabetes (T2D), metformin (Met) exhibits antitumor activity against a variety of cancer cells, with a vaguely defined mechanism. In addition, the influence of metformin on the UC and putrescine generation in colorectal cancer has remained unclear. In our study, we investigated the effect of metformin on the UC and putrescine generation of CRC in vivo and in vitro and elucidated the underlying mechanisms. In nude mice bearing HCT116 tumor xenografts, the administration of metformin inhibited tumor growth without affecting body weight. In addition, metformin treatment increased the expression of monophosphate (AMP)-activated protein kinase (AMPK) and p53 in both HCT116 xenografts and colorectal cancer cell lines and decreased the expression of the urea cycle enzymes, including carbamoyl phosphate synthase 1 (CPS1), arginase 1 (ARG1), ornithine trans-carbamylase (OTC), and ODC. The putrescine levels in both HCT116 xenografts and HCT116 cells decreased after metformin treatment. These results demonstrate that metformin inhibited CRC cell proliferation via activating AMPK/p53 and that there was an association between metformin, urea cycle inhibition and a reduction in putrescine generation.

ODC (Ornithine Decarboxylase)-Dependent Putrescine Synthesis Maintains MerTK (MER Tyrosine-Protein Kinase) Expression to Drive Resolution.

OBJECTIVE: ODC (ornithine decarboxylase)-dependent putrescine synthesis promotes the successive clearance of apoptotic cells (ACs) by macrophages, contributing to inflammation resolution. However, it remains unknown whether ODC is required for other arms of the resolution program. Approach and Results: RNA sequencing of ODC-deficient macrophages exposed to ACs showed increases in mRNAs associated with heightened inflammation and decreases in mRNAs related to resolution and repair compared with WT (wild type) macrophages. In zymosan peritonitis, myeloid ODC deletion led to delayed clearance of neutrophils and a decrease in the proresolving cytokine, IL (interleukin)-10. Nanoparticle-mediated silencing of macrophage ODC in a model of atherosclerosis regression lowered IL-10 expression, decreased efferocytosis, enhanced necrotic core area, and reduced fibrous cap thickness. Mechanistically, ODC deletion lowered basal expression of MerTK (MER tyrosine-protein kinase)-an AC receptor-via a histone methylation-dependent transcriptional mechanism. Owing to lower basal MerTK, subsequent exposure to ACs resulted in lower MerTK-Erk (extracellular signal-regulated kinase) 1/2-dependent IL-10 production. Putrescine treatment of ODC-deficient macrophages restored the expression of both MerTK and AC-induced IL-10. CONCLUSIONS: These findings demonstrate that ODC-dependent putrescine synthesis in macrophages maintains a basal level of MerTK expression needed to optimally resolve inflammation upon subsequent AC exposure. Graphic Abstract: A graphic abstract is available for this article.

BrrICE1.1 is associated with putrescine synthesis through regulation of the arginine decarboxylase gene in freezing tolerance of turnip (Brassica rapa var. rapa).

BACKGROUND: In the agricultural areas of Qinghai-Tibet Plateau, temperature varies widely from day to night during the growing season, which makes the extreme temperature become one of the limiting factors of crop yield. Turnip (Brassica rapa var. rapa) is a traditional crop of Tibet grown in the Tibet Plateau, but its molecular and metabolic mechanisms of freezing tolerance are unclear. RESULTS: Here, based on the changes in transcriptional and metabolic levels of Tibetan turnip under freezing treatment, the expression of the arginine decarboxylase gene BrrADC2.2 exhibited an accumulative pattern in accordance with putrescine content. Moreover, we demonstrated that BrrICE1.1 (Inducer of CBF Expression 1) could directly bind to the BrrADC2.2 promoter, activating BrrADC2.2 to promote the accumulation of putrescine, which was verified by RNAi and overexpression analyses for both BrrADC2.2 and BrrICE1.1 using transgenic hair root. The function of putrescine in turnip was further analyzed by exogenous application putrescine and its inhibitor DL-α-(Difluoromethyl) arginine (DFMA) under freezing tolerance. In addition, the BrrICE1.1 was found to be involved in the ICE1-CBF pathway to increase the freezing stress of turnip. CONCLUSIONS: BrrICE1.1 could bind the promoter of BrrADC2.2 or CBFs to participate in freezing tolerance of turnip by transcriptomics and targeted metabolomics analyses. This study revealed the regulatory network of the freezing tolerance process in turnip and increased our understanding of the plateau crops response to extreme environments in Tibet.

Engineering siderophores.

Siderophores have important functions for bacteria in iron acquisition and as virulence factors. In this chapter we will discuss the engineering of cyclic hydroxamate siderophores by various biochemical approaches based on the example of Shewanella algae. The marine gamma-proteobacterium S. algae produces three different cyclic hydroxamate siderophores as metabolites via a single biosynthetic gene cluster and one of them is an important key player in interspecies competition blocking swarming of Vibrio alginolyticus. AvbD is the key metabolic enzyme assembling the precursors into three different core structures and hence an interesting target for metabolic and biochemical engineering. Synthetic natural and unnatural precursors can be converted in vitro with purified AvbD to generate siderophores with various ring sizes ranging from analytical to milligram scale. These engineered siderophores can be applied, for example, as swarming inhibitors against V. alginolyticus. Here, we describe the synthesis of the natural and unnatural siderophore precursors HS[X]A and provide our detailed protocols for protein expression of AvbD, conversion of HS[X]A with the enzyme to produce ring-size engineered siderophores and secondly for a biosynthetic feeding strategy that allows to extract engineered siderophores in the milligram scale.

Macrophage Metabolism of Apoptotic Cell-Derived Arginine Promotes Continual Efferocytosis and Resolution of Injury.

Continual efferocytic clearance of apoptotic cells (ACs) by macrophages prevents necrosis and promotes injury resolution. How continual efferocytosis is promoted is not clear. Here, we show that the process is optimized by linking the metabolism of engulfed cargo from initial efferocytic events to subsequent rounds. We found that continual efferocytosis is enhanced by the metabolism of AC-derived arginine and ornithine to putrescine by macrophage arginase 1 (Arg1) and ornithine decarboxylase (ODC). Putrescine augments HuR-mediated stabilization of the mRNA encoding the GTP-exchange factor Dbl, which activates actin-regulating Rac1 to facilitate subsequent rounds of AC internalization. Inhibition of any step along this pathway after first-AC uptake suppresses second-AC internalization, whereas putrescine addition rescues this defect. Mice lacking myeloid Arg1 or ODC have defects in efferocytosis in vivo and in atherosclerosis regression, while treatment with putrescine promotes atherosclerosis resolution. Thus, macrophage metabolism of AC-derived metabolites allows for optimal continual efferocytosis and resolution of injury.

Engineering Tropane Alkaloid Production Based on Metabolic Characterization of Ornithine Decarboxylase in Atropa belladonna.

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) such as pharmaceutical hyoscyamine, anisodamine, and scopolamine, remains largely unknown. In this study, we characterized the physiological and metabolic functions of the ODC gene of Atropa belladonna (AbODC) and determined its role in TA production using metabolic engineering approaches. Feeding assays with enzyme inhibitors indicated that ODC, rather than arginine decarboxylase (ADC), plays a major role in TA biosynthesis. Tissue-specific AbODC expression analysis and ß-glucuronidase (GUS) staining assays showed that AbODC was highly expressed in secondary roots, especially in the cylinder tissue. Enzymatic assays indicated that AbODC was able to convert ornithine to putrescine, with the highest activity at pH 8.0 and 30 °C. Additionally, AbODC showed higher catalytic efficiency than other plant ODCs, as evident from the Km, Vmax, and Kcat values of AbODC using ornithine as the substrate. In A. belladonna root cultures, suppression of AbODC greatly reduced the production of putrescine, N-methylputrescine, and TAs, whereas overexpression of AbODC significantly increased the biosynthesis of putrescine, N-methylputrescine, hyoscyamine, and anisodamine. Moreover, transgenic A. belladonna plants overexpressing AbODC showed a significantly higher production of hyoscyamine and anisodamine compared with control plants. These findings indicate that AbODC plays a key role in TA biosynthesis and therefore is a valuable candidate for increasing TA production in A. belladonna.

Construction and characterization of a double mutant of Enterococcus faecalis that does not produce biogenic amines.

Enterococcus faecalis is a lactic acid bacterium characterized by its tolerance of very diverse environmental conditions, a property that allows it to colonize many different habitats. This species can be found in food products, especially in fermented foods where it plays an important role as a biopreservative and influences the development of organoleptic characteristics. However, E. faecalis also produces the biogenic amines tyramine and putrescine. The consumption of food with high concentrations of these compounds can cause health problems. The present work reports the construction, via homologous recombination, of a double mutant of E. faecalis in which the clusters involved in tyramine and putrescine synthesis (which are located in different regions of the chromosome) are no longer present. Analyses showed the double mutant to grow and adhere to intestinal cells normally, and that the elimination of genes involved in the production of tyramine and putrescine has no effect on the expression of other genes.

Effect of exogenous spermidine on floral induction, endogenous polyamine and hormone production, and expression of related genes in 'Fuji' apple (Malus domestica Borkh.).

Flower bud formation in 'Fuji' apple (Malus domestica Borkh.) is difficult, which severely constrains commercial production. Spermidine (Spd) plays an important role in floral induction, but the mechanism of its action is incompletely understood. To investigate the effect of Spd on flowering, 6-year-old 'Fuji' apple trees were treated with 1 × 10-5 mol L-1 Spd to study the responses of polyamines [putrescine (Put), Spd and spermine (Spm)], hormones [gibberellins (GA3) and abscisic acid (ABA)], and polyamine-, hormone- and flowering-related genes. Spd application promoted flowering during floral induction by increasing MdGA2ox2 (gibberellin 2-oxidase) through GA3 reduction and increasing MdNCED1 and MdNCED3 (9-cis-epoxycarotenoid dioxygenase) through ABA enrichment during 60 to 80 days after full bloom. The flowering rate as well as the expressions of flower-related genes, except for MdLEY (LEAFY), also increased, thereby promoting flowering. In addition, spraying with Spd significantly increased the contents of endogenous polyamines except for Spm in terminal buds by increasing the expressions of polyamine-associated genes. We hypothesize that the contribution of Spd to flowering is related to crosstalk among polyamines, hormone signals, and related gene expressions, which suggests that Spd participates in the apple floral induction process.

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic Escherichia coli.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T). RESULTS: Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed. CONCLUSIONS: Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

Bioactive polyamine production by a novel hybrid system comprising multiple indigenous gut bacterial strategies.

Metabolites of the intestinal microbiota are thought to be generated through metabolic pathways spanning multiple taxa of intestinal bacteria. We have previously shown that the level of putrescine, a polyamine found abundantly in the human intestinal lumen, is increased in the colonic lumen following administration of arginine and the probiotic Bifidobacterium sp.; however, the underlying mechanism remained poorly understood. We report a novel pathway for putrescine production from arginine through agmatine involving the collaboration of two bacterial groups, and triggered by environmental acidification (drop in pH to below 6.5 from neutral). This pathway comprises the acid tolerance system of Escherichia coli, representing bacteria that have an arginine-dependent acid resistance system; the energy production system of Enterococcus faecalis, representing bacteria that have an agmatine deiminase system; and the acid production system of the acid-producing bacteria, represented by Bifidobacterium spp. This pathway is unique in that it represents a relationship between the independent survival strategies of multiple bacteria.

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium.

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium. ABBREVIATIONS: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion.

Metabolomic studies identify changes in transmethylation and polyamine metabolism in a brain-specific mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and the quintessential disorder of mechanistic Target of Rapamycin Complex 1 (mTORC1) dysregulation. Loss of either causative gene, TSC1 or TSC2, leads to constitutive mTORC1 kinase activation and a pathologically anabolic state of macromolecular biosynthesis. Little is known about the organ-specific metabolic reprogramming that occurs in TSC-affected organs. Using a mouse model of TSC in which Tsc2 is disrupted in radial glial precursors and their neuronal and glial descendants, we performed an unbiased metabolomic analysis of hippocampi to identify Tsc2-dependent metabolic changes. Significant metabolic reprogramming was found in well-established pathways associated with mTORC1 activation, including redox homeostasis, glutamine/tricarboxylic acid cycle, pentose and nucleotide metabolism. Changes in two novel pathways were identified: transmethylation and polyamine metabolism. Changes in transmethylation included reduced methionine, cystathionine, S-adenosylmethionine (SAM-the major methyl donor), reduced SAM/S-adenosylhomocysteine ratio (cellular methylation potential), and elevated betaine, an alternative methyl donor. These changes were associated with alterations in SAM-dependent methylation pathways and expression of the enzymes methionine adenosyltransferase 2A and cystathionine beta synthase. We also found increased levels of the polyamine putrescine due to increased activity of ornithine decarboxylase, the rate-determining enzyme in polyamine synthesis. Treatment of Tsc2+/- mice with the ornithine decarboxylase inhibitor α-difluoromethylornithine, to reduce putrescine synthesis dose-dependently reduced hippocampal astrogliosis. These data establish roles for SAM-dependent methylation reactions and polyamine metabolism in TSC neuropathology. Importantly, both pathways are amenable to nutritional or pharmacologic therapy.

Metabolic evolution and a comparative omics analysis of Corynebacterium glutamicum for putrescine production.

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor L-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.

Effect of ornithine decarboxylase and norspermidine in modulating cell division in the green alga Chlamydomonas reinhardtii.

The extensive genetic resources of Chlamydomonas has led to its widespread use as a model system for understanding fundamental processes in plant cells, including rates of cell division potentially modulated through polyamines. Putrescine was the major polyamine in both free (88%) and membrane-bound fractions (93%) while norspermidine was the next most abundant in these fractions accounting for 11% and 6%, respectively. Low levels of diaminopropane, spermidine and spermine were also observed although no cadaverine or norspermine were detected. Ornithine decarboxylase (ODC, EC 4.1.1.17) activity was almost five times higher than arginine decarboxylase (ADC, EC 4.1.1.19) and is the major route of putrescine synthesis. The fluoride analogue of ornithine (α-DFMO) inhibited membrane associated ODC activity whilst simultaneously stimulating cell division in a dose dependent manner. Following exposure to α-DFMO the putrescine content in the cells declined while the norspermidine content increased over two fold. Addition of norspermidine to cultures stimulated cell division mimicking the effects observed using DFMO and also reversed the inhibitory effects of cyclohexylamine on growth. The results reveal that ODC is the major route to polyamine formation in the Chlamydomonas CC-406 cell-wall mutant, in contrast to the preferential ADC route reported for Chlorella vulgaris, suggesting that significant species differences exist in biosynthetic pathways which modulate endogenous polyamine levels in green algae.

Expression and distribution patterns of spermine, spermidine, and putrescine in rat hair follicle.

No expression and distribution patterns of polyamines (PAs), spermine, spermidine, and their precursor putrescine in mammalian hair follicle are available, although polyamines are known to correlate well with hair growth and epidermal tumor genesis. Immunohistochemistry (IHC) using our original two monoclonal antibodies (mAbs) ASPM-29 specific for spermine or spermidine, and APUT-32 specific for putrescine allowed us to detect immunoreactivity for polyamines in hair follicles from normal adult rats. A wide range of immunoreactivity for the total spermine and spermidine was observed in the compartments of hair follicle: The highest degree of immunoreactivity for polyamines was observed in the matrix, in the Huxley's layer, in the deeper Henle's layer, and in the cuticle of the inner root sheath/the hair cuticle, while moderate immunoreactivity existed in the lower-to-mid cortex and the companion layer, followed by lower immunoreactivity in the outer root sheath, including the bulge region and in the deeper medulla, in which the immunoreactivity was also evident in their nuclei. In addition, somewhat surprisingly, with IHC by APUT-32 mAb, we detected significant levels of putrescine in the compartments, in which the immunostaining pattern was the closely similar to that of the total spermine and spermidine. Thus, among these compartments, the cell types of the matrix, the Huxley's layer, the deeper Henle's layer, and the cuticle of the inner root sheath/the hair cuticle seem to have the biologically higher potential in compartments of anagen hair follicle, maybe suggesting that they are involved more critically in the biological event of hair growth. In addition, we noted sharp differences of immunostaining by IHCs between ASPM-29 mAb and APUT-32 mAb in the epidermis cells and fibroblast. ASPM-29 mAb resulted in strong staining in both the cell types, but APUT-32 mAb showed only very light staining in both types. Consequently, the use of the two IHCs could be extremely useful in further studies on hair cycle and epidermal tumor genesis experimentally or clinically.

Effects of probiotic bacteria on mucosal polyamines levels in dogs with IBD and colonic polyps: a preliminary study.

Spermine (SPM) and its precursor putrescine (PUT), regulated by ornithine decarboxylase (ODC) and diamino-oxidase (DAO), are polyamines required for cell growth and proliferation. Only a few studies have investigated the anti-inflammatory and tumour inhibitory properties of probiotics on mucosal polyamine levels. We investigated the effects of a high concentration multistrain probiotic for human use on colonic polyamine biosynthesis in dogs. Histological sections (inflammatory bowel disease, n=10; polyposis, n=5) were assessed after receiving 112 to 225×109 lyophilised bacteria daily for 60 days at baseline (T0) and 30 days after treatment end (T90). Histology scores, expression of PUT, SPM, ODC and DAO, and a clinical activity index (CIBDAI) were compared at T0 and T90. In polyps, cellular proliferation (Ki-67 expression), and apoptosis (caspase-3 protein expression) were also evaluated. After treatment, in inflammatory bowel disease significant decreases were observed for CIBDAI (P=0.006) and histology scores (P<0.001); PUT, SPM and ODC expression increased (P<0.01). In polyps, a significant decrease in polyamine levels, ODC activity, and Ki-67, and a significant increase in caspase-3 positivity and DAO expression (P=0.005) was noted. Our results suggest potential anti-proliferative and anti-inflammatory effects of the probiotic mixture in polyps and inflammation, associated with reduced mucosal infiltration and up-regulation of PUT, SPM, and ODC levels.

[Low-molecular-weight regulators of biogenic polyamine metabolism affect cytokine production and expression of hepatitis С virus proteins in Huh7.5 human hepatocarcinoma cells].

Hepatitis C virus (HCV) induces the expression of the genes of proinflammatory cytokines, the excessive production of which may cause cell death, and contribute to development of liver fibrosis and hepatocarcinoma. The relationship between cytokine production and metabolic disorders in HCV-infected cells remains obscure. The levels of biogenic polyamines, spermine, spermidine, and their precursor putrescine, may be a potential regulator of these processes. The purpose of the present work was to study the effects of the compounds which modulate biogenic polyamines metabolism on cytokine production and HCV proteins expression. Human hepatocarcinoma Huh7.5 cells have been transfected with the plasmids that encode HCV proteins and further incubated with the following low-molecular compounds that affect different stages of polyamine metabolism: (1) difluoromethylornithine (DFMO), the inhibitor of ornithine decarboxylase, the enzyme that catalyzes the biosynthesis of polyamines; (2) N,N'-bis(2,3-butane dienyl)-1,4-diaminobutane (MDL72.527), the inhibitor of proteins involved in polyamine degradation; and (3) synthetic polyamine analog N^(I),N^(II)-diethylnorspermine (DENSpm), an inducer of polyamine degradation enzyme. The intracellular accumulation and secretion of cytokines (IL-6, IL-1ß, TNF-α, and TGF-ß) was assessed by immunocytochemistry and in the immunoenzyme assay, while the cytokine gene expression was studied using reverse transcription and PCR. The effects of the compounds under analysis on the expression of HCV proteins were analyzed using the indirect immunofluorescence with anti-HCV monoclonal antibodies. It has been demonstrated that, in cells transfected with HCV genes, DFMO reduces the production of three out of four tested cytokines, namely, TNF-α and TGF-ß in cells that express HCV core, Е1Е2, NS3, NS5A, and NS5B proteins, and IL-1ß in the cells that express HCV core, Е1Е2, and NS3 proteins. MDL72527 and DENSpm decreased cytokine production to a lesser extent. Incubation with DFMO led to a 28-32% decrease in the number of cells expressing NS5B or NS5A, both of which are key components of the HCV replication complex. The results obtained in the work indicate that a further detailed study of the antiviral activity of DFMO is required in order to assess its potential as an anti-hepatitis C therapeutic agent.

Dual functioning of plant arginases provides a third route for putrescine synthesis.

Two biosynthetic routes are known for putrescine, an essential plant metabolite. Ornithine decarboxylase (ODC) converts ornithine directly to putrescine, while a second route for putrescine biosynthesis utilizes arginine decarboxylase (ADC) to convert arginine to agmatine, and two additional enzymes, agmatine iminohydrolase (AIH) and N-carbamoyl putrescine aminohydrolase (NLP1) to complete this pathway. Here we show that plants can use ADC and arginase/agmatinase (ARGAH) as a third route for putrescine synthesis. Transformation of Arabidopsis thaliana ADC2, and any of the arginases from A. thaliana (ARGAH1, or ARGHA2) or the soybean gene Glyma.03g028000 (GmARGAH) into a yeast strain deficient in ODC, fully complemented the mutant phenotype. In vitro assays using purified recombinant enzymes of AtADC1 and AtARGAH2 were used to show that these enzymes can function in concert to convert arginine to agmatine and putrescine. Transient expression analysis of the soybean genes (Glyma.06g007500, ADC; Glyma.03g028000 GmARGAH) and the A. thaliana ADC2 and ARGAH genes in leaves of Nicotiana benthamiana, showed that these proteins are localized to the chloroplast. Experimental support for this pathway also comes from the fact that expression of AtARGAH, but not AtAIH or AtNLP1, is co-regulated with AtADC2 in response to drought, oxidative stress, wounding, and methyl jasmonate treatments. Based on the high affinity of ARGAH2 for agmatine, its co-localization with ADC2, and typically low arginine levels in many plant tissues, we propose that these two enzymes can be major contributors to putrescine synthesis in many A. thaliana stress responses.